Morphokinetic analysis of cleavage stage embryos and assessment of specific gene expression in cumulus cells independently predict human embryo development to expanded blastocyst: a preliminary study.

To assess whether morphokinetic features at the cleavage stage together with specific gene expression in cumulus cells (CCs) may be used to predict whether human embryos are able to achieve the expanded blastocyst stage on day 5. Eighty-one embryos were cultured using the Geri plus® time-lapse system. Twenty-seven embryos progressing to the expanded blastocyst stage (BL group) were compared with thirty-five embryos showing developmental arrest (AR group) and nineteen reaching the stage of early or not fully expanded blastocyst (nBL group). The analyzed morphokinetic variables were pronuclear appearance (tPNa), pronuclear fading (tPNf), and completion of cleavage to two, three, four, and eight cells (t2, t3, t4, and t8). CCs were analyzed by RT-qPCR for bone morphogenetic protein 15 (BMP15), cytochrome c oxidase subunit II (COXII), ATP synthase subunit 6 (MT-ATP6), connexin 43 (Cx43), and heme oxygenase-1 (HO-1). Embryos of BL group showed a significantly faster kinetic. BMP15, COXII, and MT-ATP6 mRNA expression was significantly higher in CCs of BL group embryos, whereas Cx43 and HO-1 mRNA levels were higher in AR group. Kinetic parameters and gene expression were not significantly different between either the BL and nBL groups or the AR and nBL groups. ROC curves showed that the most predictive cut-offs were t2 < 26.25 for morphokinetics and COXII > 0.3 for gene expression. Multivariable logistic regression analysis showed that morphokinetic variables and gene expression were both valuable, independent predictors of embryo development to expanded blastocyst. Our results suggest the possibility of developing integrated prediction models for early embryo selection at the cleavage stage.

Removing the zona pellucida can decrease cytoplasmic fragmentations in human embryos: a pilot study using 3PN embryos and time-lapse cinematography.

PURPOSE: The aim of this study was to establish a new method of decreasing cytoplasmic fragmentation in early-stage human embryos. METHODS: The zona pellucida (ZP) of abnormally-fertilized oocytes (zygotes with three pronuclei (3PN)), which were donated by patients, was removed at the pronuclear stage. ZP-free embryos were observed in a time-lapse imaging and culturing system in order to examine developmental morphology and embryonic quality. RESULTS: Based on a modification of Veeck's criteria, 47 of 69 ZP-free 3PN embryos (68.1%) showed fragmentation of less than 20% of the total volume of cytoplasm at the first cleavage (grades 1 and 2), 17 (24.6%) showed 20-40% cytoplasmic fragments (grade 3), and only 5 (7.2%) showed more than 40% fragments (grade 4). These results suggest that the rate of fragmentation is decreased by ZP removal before the first cleavage, compared with normal (ZP-intact) 3PN and 2-pronuclear/2-polar body embryos. CONCLUSIONS: This study revealed that the ZP is not always necessary for normal development after the pronuclear stage because the ZP-free embryos studied herein developed normally, maintained their cell adhesion well, and showed a decreased rate of fragmentation. This innovative culture system might provide the major breakthrough needed for patients who have difficulty obtaining good-quality embryos.

The cleavage program in the 2d cell lineage of Tubifex embryos.

Early development in clitellate annelids is characterized by a highly stereotyped sequence of unequal, spiral cleavages. Cell 2d (i.e., the second micromere of the D quadrant) in the oligochaete Tubifex tubifex also undergoes an evolutionarily conserved sequence of cell division to produce four bilateral pairs of ectodermal teloblasts that act as embryonic stem cells. This study was conducted to characterize each of the 15 rounds of cell division that occur in the 2d cell lineage in this clitellate. After its occurrence, cell 2d undergoes three rounds of highly unequal divisions, giving off the first smaller daughter cell toward the posterior right of the larger daughter cell, the second cell toward the posterior left, and the third cell toward the anterior side of the cell; the larger daughter cell that results from the third division (i.e., the great-granddaughter cell of 2d) then divides equally into a bilateral pair of NOPQ proteloblasts. Cell NOPQ on either side of the embryo undergoes 11 rounds of cell division, during which ectoteloblasts N, Q, and O/P are produced in this order. After its appearance, NOPQ undergoes highly unequal divisions twice cutting off the smaller cells toward the anterior end of the embryo and then divides almost equally into ectoteloblast N and proteloblast OPQ. After its appearance, OPQ undergoes highly unequal divisions twice giving off the first smaller cell toward the anterior and the second smaller cell toward the posterior of the embryo and then divides almost equally into ectoteloblast Q and proteloblast OP. Finally, OP undergoes highly unequal division four times after its birth budding off the smaller cells toward the anterior and then cleaves equally into ectoteloblasts O and P. In the unequally dividing cells of the 2d cell lineage, the mitotic apparatus (MA), which forms at the cell's center, moves eccentrically toward the cortical site where the smaller cell will be given off. The moving MA is oriented perpendicular to the surface it approaches, and its peripheral pole becomes closely associated with the cell cortex. In contrast, the MA involved in the equal divisions remains in the cell center throughout mitosis. The key features of the cleavage program in the 2d cell lineage are discussed in light of the present observations. The mechanical aspects of unequal cleavage in the 2d cell lineage and the modes of specification of MA orientation are discussed. A comparison of the cleavage mode in the 2d cell lineage is also performed among six selected clitellate annelid species.

Irregular cleavage of early preimplantation human embryos: characteristics of patients and pregnancy outcomes.

PURPOSE: This is a retrospective analysis of the morphokinetics, prevalence, and implantation potential of embryos with irregular first and second cleavages as identified by time-lapse microscopy. METHODS: The study included 253 women who underwent 387 assisted reproduction treatments with intracytoplasmic sperm injection (ICSI). Each patient was assigned to one of three groups based on embryo cleavage results. In group I, one to two embryos per cycle showed irregular cleavage; group II, at least three embryos with abnormal cleavage; and in group III (the control group), all embryos cleaved normally. The number of embryos that cleaved from 1 to ≥3 cells or from 2 to ≥5 cells for each patient was recorded. Their prevalence and association with women's characteristics and pregnancy outcome were evaluated. RESULTS: The prevalence of irregular cleavage was 15.6 % among 1772 ICSI embryos. In 101 cycles, 1-2 embryos per cycle showed irregular cleavage (group I). In 32 cycles, at least 3 embryos showed abnormal cleavage (group II). In 254 cycles, all embryos cleaved normally (group III). The average age of the women in group II was significantly lower in comparison with groups I and III (32.5 ± 4.2 vs. 35.1 ± 4.9 and 35.5 ± 5.1, respectively, p < 0.02). In comparison of groups I and II, the odds ratio for ≥3 embryos with irregular cleavage in women younger than 35 was 3.48 (95 % CI, 1.28 to 9.46). Embryos with irregular cleavage were transferred in 16 women. Three live births were achieved following the transfer of single blastocysts derived from embryos with irregular cleavage from two to five cells. CONCLUSIONS: Early embryos with irregular cleavage are significantly more prevalent in younger women. When these embryos develop to the blastocyst stage, they may have normal implantation potential, leading to the birth of healthy babies.

Cellular analysis of cleavage-stage chick embryos reveals hidden conservation in vertebrate early development.

Birds and mammals, phylogenetically close amniotes with similar post-gastrula development, exhibit little conservation in their post-fertilization cleavage patterns. Data from the mouse suggest that cellular morphogenesis and molecular signaling at the cleavage stage play important roles in lineage specification at later (blastula and gastrula) stages. Very little is known, however, about cleavage-stage chick embryos, owing to their poor accessibility. This period of chick development takes place before egg-laying and encompasses several fundamental processes of avian embryology, including zygotic gene activation (ZGA) and blastoderm cell-layer increase. We have carried out morphological and cellular analyses of cleavage-stage chick embryos covering the first half of pre-ovipositional development, from Eyal-Giladi and Kochav stage (EGK-) I to EGK-V. Scanning electron microscopy revealed remarkable subcellular details of blastomere cellularization and subgerminal cavity formation. Phosphorylated RNA polymerase II immunostaining showed that ZGA in the chick starts at early EGK-III during the 7th to 8th nuclear division cycle, comparable with the time reported for other yolk-rich vertebrates (e.g. zebrafish and Xenopus). The increase in the number of cell layers after EGK-III is not a direct consequence of oriented cell division. Finally, we present evidence that, as in the zebrafish embryo, a yolk syncytial layer is formed in the avian embryo after EGK-V. Our data suggest that several fundamental features of cleavage-stage development in birds resemble those in yolk-rich anamniote species, revealing conservation in vertebrate early development. Whether this conservation lends morphogenetic support to the anamniote-to-amniote transition in evolution or reflects developmental plasticity in convergent evolution awaits further investigation.

Morphokinetic parameters of ICSI tripronucleated embryos observed using time lapse.

Time-lapse analysis of tripronucleated zygotes obtained in ICSI cycles showed that 75.4% cleaved into embryos. These embryos subsequently demonstrated slower developmental kinetics than normally fertilized embryos.

Blastocoel-spanning filopodia in cleavage-stage Xenopus laevis: Potential roles in morphogen distribution and detection.

In the frog Xenopus laevis, dorsal-ventral axis specification involves cytoskeleton-dependent transport of localized transcripts and proteins during the first cell cycle, and activation of the canonical Wnt pathway to locally stabilize translated beta-catenin which, by as early as the 32-cell stage, commits nuclei in prospective dorsal lineages to the subsequent expression of dorsal target genes. Maternal ligands important for activating this dorsal-specific signaling pathway are thought to interact with secreted glypicans and coreceptors in the blastocoel. While diffusion between cells is generally thought of as sufficient to accomplish the distribution of secreted maternal ligands to their appropriate targets, signaling may also involve other potential mechanisms, including direct transfer of morphogens via membrane-bounded entities, such as argosomes, exosomes, or even filopodia. In Xenopus, the blastocoel-facing, basolateral surfaces where signaling interactions ostensibly take place have not been previously examined in detail. Here, we report that the cleavage-stage blastocoel is traversed by hundreds of extremely long cellular protrusions that maintain long-term contacts between nonadjacent blastomeres during expansion of the interstitial space in early embryogenesis. The involvement of these protrusions in early embryonic patterning is suggested by the discoveries that (a) they fragment into microvesicles, whose resorption facilitates considerable exchange of cytoplasm and membrane between blastomeres; and (b) they are active in caveolar endocytosis, a prerequisite for ligand-receptor signaling.

Early compaction at day 3 may be a useful additional criterion for embryo transfer.

PURPOSE: The reduction of the number of embryos transferred while maintaining a satisfactory rate of pregnancy (PR) with in vitro fertilization calls for a refined technique of embryonic selection. This prospective study investigates the significance of early embryonic compaction at day 3 as a marker of the chances of implantation. METHODS: We examined 317 transfers and their outcome involving 509 embryos including 91 compacted embryos. RESULTS: Early compaction seems linked with the ovarian response to stimulation and embryonic quality. The PR is significantly increased when the embryonic cohort contains at least one compacted embryo (44% versus 29.5%, p = 0.01), and when at least one compacted embryo is transferred (44% versus 31%, p < 0.05). The analysis of our single embryo transfers shows that the implantation rates are significantly better for compacted embryos (50% versus 30%, p < 0.05) (OR 2.98; CI 1.02-5.28). CONCLUSION: Thus, early compaction, sometimes observed at day 3, may serve as a useful additional criterion for selecting the embryos transferred.

[A comparative study of fluorescent patterns in the nuclei of early mouse embryos using antibodies against different domains of the actin molecule].

In this work, new data on the peculiarities of actin immunofluorescent detection in 2-cell mouse embryos using antibodies against C- and N-terminal domains are presented. We studied the distribution of nuclear actin after artificial suppression of transcription and after enzymatic digestion of DNA. The visual observations were supplemented with morphometric analyses of confocal images. In both experimental groups (treated by transcription inhibitors or DNase) the reliable increasing of fluorescence intensity is revealed when antibody against C-, but not against N-terminal domain of actin was used. These finding allows to suppose that antibody against C-terminal domain of actin is suitable with a high efficiency for immunofluorescent studies on the nuclei of cleavage mouse embryo.

Breakage-fusion-bridge cycles leading to inv dup del occur in human cleavage stage embryos.

Recently, a high incidence of chromosome instability (CIN) was reported in human cleavage stage embryos. Based on the copy number changes that were observed in the blastomeres it was hypothesized that chromosome breakages and fusions occur frequently in cleavage stage human embryos and instigate subsequent breakage-fusion-bridge cycles. In addition, it was hypothesized that the DNA breaks present in spermatozoa could trigger this CIN. To test these hypotheses, we genotyped both parents as well as 93 blastomeres from 24 IVF embryos and developed a novel single nucleotide polymorphism (SNP) array-based algorithm to determine the parental origin of (aberrant) loci in single cells. Paternal as well as maternal alleles were commonly rearranged in the blastomeres indicating that sperm-specific DNA breaks do not explain the majority of these structural variants. The parent-of-origin analyses together with microarray-guided FISH analyses demonstrate the presence of inv dup del chromosomes as well as more complex rearrangements. These data provide unequivocal evidence for breakage-fusion-bridge cycles in those embryos and suggest that the human cleavage stage embryo is a major source of chromosomal disorders.

Severe teratozoospermia and its influence on pronuclear morphology, embryonic cleavage and compaction.

BACKGROUND: Fertilization, cell division and embryo development depend on genomic contributions from male and female gametes. We hypothesize that teratozoospermic sperm influences early embryo development and embryo compaction. METHODS: We conducted a retrospective analysis of embryos derived from intracytoplasmic sperm injection (ICSI) cycles. Two hundred thirty-five consecutive ICSI cycles were included in the study; all treatment was provided at the Cleveland Clinic Fertility Center. Patient cycles were divided by sperm morphology based on Kruger's strict criteria: Group A, embryos where teratozoospermic sperm (0-2% normal) were used for ICSI and Group B, embryos where dysmorphic sperm (5-13% normal) were used for ICSI. All cycles analyzed were of patients doing day 3 embryo transfers. Outcome measures assessed included pronuclear (PN) pattern, syngamy, early cleavage, cell number, rate of compaction and blastulation of embryos left in culture and not transferred on day 3. RESULTS: A total of 1762 embryos were analyzed. PN patterns were similar in Group A and Group B embryos. No differences were noted in syngamy, cleavage, cell number or blastulation rate. Studying the development of embryos in culture after day 3 transfer revealed a difference in the timeline for compaction. By day 4, 25% of Group A embryos had compacted compared to 36% in Group B (P = 0.0007). There was no difference found between Group A and Group B embryos in regards to blastulation. CONCLUSIONS: We did not find an association between sperm morphology and clinical outcomes. The impact of teratozoospermia may be masked in ICSI cycles where fertilization, implantation rate and clinical pregnancy rate are the primary outcome measures. However, by examining the timeline of development, we were better able to discern a potential paternal effect at critical transition points from fertilization through activation.

Human zygote morphological indicators of higher rate of arrest at the first cleavage stage.

A little studied aspect of developmental arrest (DA) in ART is zygote arrest (ZA). Etiologically, blockage at the first cleavage stage includes molecular and chromosomal anomalies, some of which manifest morphologically. Given considerations on embryo culture, transfer and cryopreservation, optimal zygote selection is very important. The aim of this study was to ascertain whether zygote morphological features were indicators of increased ZA. In this study we performed a prospective, observational study of 2105 zygotes obtained from consecutive patients who were undergoing IVF/ICSI treatment, of which 43 (2%) suffered ZA. Morphological features observed under the inverted microscope were qualitatively categorized: pronuclear size, nucleolar precursor bodies (NPB) alignment, light and dark halos, polar body placement and fragmentation observed at 16-18 h post-insemination. We compared these features in blocked versus cleaved zygotes at 48 h and found significant correlations (p < 0.05) between ZA and three features: the absence of a light halo (p = 0.001), the absence of a dark halo (p < 0.005), and non-aligned NPB (p < 0.05). We can say that certain morphological features are indicators of significantly increased zygote arrest. These findings may be of utility for optimal zygote selection and culture strategies, especially in countries under restrictive conditions.

Maternal diabetes increases apoptosis in mice oocytes, not 2-cell embryos.

Apoptosis may be closely involved in diabetes-induced embryonic malformations. We aimed to investigate the occurrence of apoptosis at an early stage of development, in oocytes and 2-cell embryos of streptozotocin-induced diabetic mice and nondiabetic mice. Diabetic mouse ovarian sections stained with hematoxylin and eosin showed reduced number of growing follicles and delayed oocyte development. Annexin V-positive oocytes were higher in number in diabetic mice than in nondiabetic mice. Quantitative RT-PCR and immunofluorescence analysis revealed the expression of Bax and caspase-3 significantly higher in diabetic than nondiabetic oocytes. In contrast, 2-cell embryos of diabetic and nondiabetic mice showed no annexin V-positive staining. Bax expression was elevated in diabetic 2-cell embryos, but caspase-3 expression did not significantly differ between diabetic and nondiabetic 2-cell embryos. Electron microscopy revealed increased number of swollen mitochondria in diabetic 2-cell embryos. These results suggested that maternal diabetes might increase oocyte apoptosis by a Bax-caspase-3 pathway to play a role in embryonic malformations by delayed oocyte development. Development of 2-cell embryos might be adversely affected by maternal diabetes, but not through Bax-regulated caspase-3 apoptotic pathway.

[Last morphological selection]. / Implantation et choix de l'embryon. Sélection morphologique ultime.

Selection criteria for embryo transfer is an essential step in ART. Evaluation of pronuclear morphology, evaluation of zygote, embryo cleavage, quality of blastomeres predict the viability of embryos. Multinucleation in cleavage stage embryos is associated with a lower implantation and pregnancy rate.

Evaluation of the safety of time-lapse observations for human embryos.

PURPOSE: To assess the effects of light from an integrated optical microscope and evaluate the safety of time-lapse observations using a built-in microscope incubator. METHODS: We prospectively compared the fertilization rate and embryonic morphology after intracytoplasmic sperm injection between embryos cultured with time-lapse observations every 15 min in an incubator with an integrated optical microscope and embryos with intermittent observations (once a day) in conventional incubators. RESULTS: No significant differences were observed in the fertilization rate (57.5% vs. 57.5%) or the rate of excellent-good cleavage embryos (36.0% vs. 36.0%). CONCLUSIONS: These results suggest that time-lapse observations using an incubator with an integrated optical microscope may therefore be safely utilized in clinical practice.

Automatic user-independent zona pellucida imaging at the oocyte stage allows for the prediction of preimplantation development.

OBJECTIVE: To analyze whether a change in three-dimensional structure of the zona pellucida could indicate suboptimal gamete quality. DESIGN: Prospective study. SETTING: Women's general hospital. PATIENT(S): A total of 72 patients who gave informed consent. INTERVENTION(S): The birefringence of all oocytes was prospectively analyzed with an automatic user-independent polarization microscopy imaging system. MAIN OUTCOME MEASURE(S): Birefringence of the inner zona layer, preimplantation development, implantation, and pregnancy. RESULT(S): In approximately one third of all gametes (244/712), the system's automatic detection of the inner zona layer did not succeed. This phenomenon was a negative predictor of compaction (P<0.01), blastulation (P<0.001), and pregnancy (P<0.001). In cases of successful zona imaging, the score based on the birefringence of the inner zona layer was a strong predictor of blastocyst formation but not of embryo quality or pregnancy (P>0.05). Interestingly, antagonist protocol resulted in lower zona scores as compared with the long protocol (P<0.05). CONCLUSION(S): Combining the information from both undetected and detected oocytes, zona imaging was a helpful tool in oocyte selection. This knowledge might further help to reduce both the time in culture and the number of concepti considered for transfer.

Evaluation of day one embryo quality and IVF outcome--a comparison of two scoring systems.

BACKGROUND: The aim of our retrospective study was to compare the clinical usefulness of two non-invasive embryo scoring systems based either on a simplified pronuclear morphology of the zygote or on early cleavage rate, as well as their combination, for the selection of embryos with the best implantation potential in embryo transfer (ET). METHODS: Over a period of five years, the quality of 2708 embryos from 364 IVF cycles in women under the age of 39 years was assessed using these scoring systems in a university assisted reproduction centre. ET was always performed on day 3 of cultivation. The outcome of ETs of 702 embryos scored in the respective systems or their combination was retrospectively analyzed in terms of biochemical (bPR) and clinical pregnancy rates (cPR) and implantation rate (IR). Mann-Whitney U test and t-test for differences between relative values were used, p < 0.05 was considered statistically significant. RESULTS: There was no difference in outcome parameters in 109 cycles where only Pattern "0" zygotes, according to our simplified pronuclear morphology classification, were transferred and 140 cycles where only "other" pattern zygotes were transferred, regardless of their cleavage rate. On the contrary, significantly greater cPR and IR (p = 0.003 and p = 0.006, respectively) were achieved in 120 cycles where only early cleavage (EC) embryos were transferred compared with 152 cycles where only non early cleavage (NEC) embryos were transferred regardless of their pronuclear morphology. The best outcome in terms of cPR (56%) and IR (43%) was found in 50 cycles when Pattern "0" and EC embryos only were used for transfer. CONCLUSION: The results indicate that early cleavage is a better independent marker of implantation potential than zygote morphology. The best outcome can be achieved if both embryo scoring systems are used jointly and the embryo is classified as EC and Pattern "0".

Cleavage pattern, gastrulation, and neurulation in the appendicularian, Oikopleura dioica.

The appendicularian, Oikopleura dioica is a chordate. Its life cycle is extremely short--approximately 5 days--and its tadpole shape with a beating tail is retained throughout entire life. The tadpole hatches after 3 h of development at 20 degrees C. Here, we describe the cleavage pattern and morphogenetic cell movements during gastrulation and neurulation. Cleavage showed an invariant pattern. It is basically bilateral but also shows various minor left-right asymmetries starting from the four-cell stage. We observed two rounds of unequal cleavage of the posterior-vegetal B-line cells at the posterior pole. The nature of the unequal cleavages is reminiscent of those in ascidian embryos and suggests the presence of a centrosome-attracting body, a special subcellular structure at the posterior pole. The representation of the cell division pattern in this report will aid the identification of each cell, a prerequisite for clarifying the gene expression patterns in early embryos. Gastrulation started as early as the 32-cell stage and progressed in three phases. By the end of the second phase at the 64-cell stage, every vegetal cell had ingressed into the embryo, and animal cells had covered the entire embryo by epiboly. There was no archenteron formation. In the anterior region, eight A-line cells were aligned as a 2x4 array along the anterior-posterior axis and become internalized during the 64-cell stage. This process was considered to correspond to neurulation. The simple and accelerated development of Oikopleura, nevertheless giving rise to a conserved chordate body plan, is advantageous for studying developmental mechanisms using molecular and genetic approaches and makes this animal the simplest model organism in the phylum Chordata.

Membrane dynamics of cleavage furrow closure in Xenopus laevis.

Epithelial membrane polarity develops early in Xenopus development, with membrane inserted along the earliest cleavage furrows by means of localized exocytosis. The added surface constitutes a new basolateral domain important for early morphogenesis. This basolateral surface becomes isolated from the outside by furrow closure, a zippering of adjacent apical-basolateral margins. Time-lapse microscopy of membrane-labeled embryos revealed two distinct kinds of protrusive activity in furrow closure. Early in furrowing, protrusive activity was associated with purse-string contractility along the apical-basolateral margins. Later in furrow progression, a basolateral protrusive zone developed entirely within the new membrane domain, with long motile filopodia extending in contractile bands from the exposed surfaces. Filopodia interacting with opposing cell surfaces across the cleavage furrow appeared to mediate blastomere-blastomere adhesion, contact spreading and lamellipodial protrusion. Interference with these dynamic activities prevented furrow closure, indicating a basic role for both marginal and basolateral protrusive activities in early embryogenesis.

[Cytochalasin B induced changes in cytoplasmic Na+/K+ balance in 2-cell mouse embryo].

This work was performed to study changes in intracellular elemental (Na/K) concentrations caused by Cytochalasin B in two-cell mouse embryo using Electron Probe Microanalysis. The presence of Cytochalasin B is required to transfer a somatic cell nuclear into an early embryo cell. The direct effect of this chemical is cytoskeleton transformation, which would be able to cause the increase of potassium channel activity resulting in cytoplasmic Na/K imbalance. In our study Cytochalasin B was shown to decrease the intracellular sodium concentration. The Na/K balance in the cytoplasm of mouse embryos reverted to its intact level after treatment them with Cytochalasin B free Dulbecco's solution. Possible mechanisms responsible for the changes in the intracellular sodium concentration observed in the embryo cells are discussed.